WO2012002745A2 - Formation d'un complexe avec le facteur viia au moyen d'un fragment d'immunoglobuline - Google Patents

Formation d'un complexe avec le facteur viia au moyen d'un fragment d'immunoglobuline Download PDF

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WO2012002745A2
WO2012002745A2 PCT/KR2011/004796 KR2011004796W WO2012002745A2 WO 2012002745 A2 WO2012002745 A2 WO 2012002745A2 KR 2011004796 W KR2011004796 W KR 2011004796W WO 2012002745 A2 WO2012002745 A2 WO 2012002745A2
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facviia
immunoglobulin
complex
region
peptidyl polymer
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PCT/KR2011/004796
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English (en)
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WO2012002745A3 (fr
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Dae Hae Song
Sung In Lim
Chang Hwan Kim
Sung Kap Hong
Dae Seong Im
Se Chang Kwon
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Hanmi Holdings Co., Ltd
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Priority to JP2013518254A priority Critical patent/JP2013533875A/ja
Priority to CN2011800325807A priority patent/CN103025358A/zh
Priority to US13/807,572 priority patent/US20130095090A1/en
Priority to EP11801151.9A priority patent/EP2588142A2/fr
Publication of WO2012002745A2 publication Critical patent/WO2012002745A2/fr
Publication of WO2012002745A3 publication Critical patent/WO2012002745A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to a blood coagulation complex for use in long-acting formulations of factor VIIa (FacVIIa). More particularly, the present invention relates to a blood coagulation complex in which FacVIIa, a non-peptidyl polymer and the immunoglobulin Fc region are held together by covalent bonds, so that the serum half-life is significantly enhanced, the blood clotting function is maintained, and the compliance of the role behavior of patients is dramatically improved. Also, the present invention is concerned with a method for preparing the blood coagulation factor complex.
  • Hemophilia A is caused by blood coagulation factor VIII deficiency and is the most common form of the disorder, accounting for 80% of hemophilia cases.
  • Hemophilia B is a disorder involving a lack of blood coagulation factor XI, and comprises approximately 20% of hemophilia cases.
  • FacVIIa is an active form of FacVII.
  • FacVII produced by the liver, is an enzyme composed of 406 amino acids, with the gamma-carboxylated glutamic acid at position 10, N-glycosylated asparagines at positions 145 and 322, and O-glycosylated serines at positions 52 and 60. It has two EGF-like domains and one serine protease domain and is activated by the bond cleavage between arginine at position 152 and isoleucine at position 153, which results in the exposure of an active site in a heavy chain. In this process, FacVII which is a single chain bonded to a light chain and heavy chain together is converted to FacVIIa which has a two-chain structure with separated light and heavy chains.
  • FacVIIa acts in the subsidiary pathway of the blood coagulation mechanism, without producing antibodies, so that it can be administered at a high dose.
  • FacVIIa is applicable for the treatment of both hemophilia A and B, and starts to be used as an alternative to conventional therapeutics for hemophilia due to its ability to be used safely in terms of antibody production and high dose administration.
  • the production of antibodies in response to the factors given to hemophiliacs makes it difficult to apply other coagulation factors tha FacVIIa.
  • FacVIIa although not causing the production of antibodies thereto, has the shortest serum half-life among the various blood coagulation factors. Thus, FacVIIa has to be administered not only frequently, causing pain to patients, but also in a large amount, imparting an economic burden to patients. To overcome these disadvantages, FacVIIa should be formulated into a long-acting form anticipated to be used for the prophylaxis of hemophilia, but not only as a supplemental factor upon hemorrhage.
  • rVIIa-FP CSL Behring
  • albumin is fused to the C-terminus of FacVIIa is in the pre-clinical phase and was found to have a serum half-life that was a 6.7-fold increase compared to native FacVIIa in rats.
  • a serum half-life as short of 4.38 hours is very short so that it is still insufficient for it to be effectively used in the prophylaxis and treatment of hemophilia.
  • PEGLip-FVIIa (Omri), a pegylated liposome formulation of FacVIIa, is in the pre-clinical phase and has a serum half-life only twice as long as that of native FacVIIa.
  • MAXY-VII (Bayer/Maxygen) and NN7128(Novo/Neose) are Factor VII products both of which have prolonged serum half lives by Gla domain mutation and hyperglycosylation and by 40K PEGylation, respectively, but in the progress of phase 1 and 2 study for each their serum half-life is five times longer than that of native FacVII, which is still insufficient for the prophylaxis and treatment of hemophilia.
  • FacVIIa complex which allows the serum half-life of FacVIIa to be prolonged with the maintenance of in vivo activity in a relatively high level, thus exerting excellent coagulation functionality, a long-acting formulation comprising the same, and a method for the preparation thereof.
  • the FacVIIa complex of the present invention guarantees the in vivo activity of FacVIIa and significantly enhances the serum half live of FacVIIa, so that it is useful for developing long-acting FacVIIa formulations which can the compliance of role behaviors of patients whose blood does not coagulate.
  • FIG. 1 is a plot showing changes in the blood levels of FacVIIa and immunoglobulin Fc-PEG-FacVIIa with time in SD rats.
  • FIG. 2 is a graph showing results of a comparative in vitro efficacy test of Novoseven, FacVIIa, and immunoglobulin Fc-PEG-FacVIIa.
  • FIG. 3 is a graph showing results of a comparative in vivo efficacy test of Novoseven, FacVIIa, and immunoglobulin Fc-PEG-FacVIIa.
  • the present invention addresses a FacVIIa complex in which FacVIIa is linked to an immunoglobulin Fc region via a non-peptidyl polymer.
  • FacVIIa refers to an active form of coagulation factor VII.
  • the FacVIIa complex of the present invention is prepared through the activation of a FacVII complex.
  • a long-acting complex is prepared from FacVII, a non-peptidyl polymer and an immunoglobulin Fc region and then allowed to undergo an activation process to form a FacVIIa complex comprised of FacVIIa, a non-peptidyl polymer and an immunoglobulin Fc region, during which in vivo activity of the complex is increased and it becomes structurally more homogeneous.
  • the activation process of converting the FacVII complex into the FacVIIa complex may include, but is not limited to, an on-column activation process and an in-solution activation process. With a preference, an on-column activation was used for FacVII complex in this invention.
  • FacVII complex is immobilized onto an anionic exchange column and then subjected to “autoactivation” without particular additives.
  • the in-solution activation process requires various factors, for example, calcium ion concentration, pH, temperature and FacVII concentration, for the activation of FacVII.
  • the FacVIIa of the present invention is an activated peptide of FacVII that is involved in the subsidiary pathway of the blood coagulation mechanism.
  • the peptide are an active form of native FacVII, FacVIIa agonists, precursors, derivatives, fragments, and variants.
  • FacVIIa agonist refers to a substance that exhibits the same biological activity as that of FacVIIa irrespective of the structure of FacVIIa.
  • FacVIIa derivative refers to a peptide that has the function of regulating blood coagulation in vivo, with at least 80% amino acid sequence homology to native FacVIIa and that may be modified at some amino acid residues by chemical substitution (e.g., alpha-methylation, alpha-hydroxylation), deletion (e.g., deamination) or decoration (e.g., N-methylation).
  • FacVIIa derivative refers to a peptide in which one or more amino acid residues are added to or deleted from the amino acid sequence of FacVIIa and that has blood coagulation activity in vivo.
  • the added amino acid residues may be non-natural amino acids (e.g., D-amino acid).
  • FacVIIa variant refers to a peptide that is different in amino acid sequence by one or more amino acids from FacVIIa and that has blood coagulation activity in vivo.
  • a peptide having a combination of the properties thereof for example, a peptide that is different in amino acid sequence by one or more amino acid residues and is deaminated at its N-terminal amino acid may be used so long as it has a blood clotting function.
  • the FacVIIa complex of the present invention is composed of a non-peptidyl polymer, an immunoglobulin Fc region and FacVIIa, with a linkage between one end of the non-peptidyl polymer and the immunoglobulin Fc region and between the other end of the non-peptidyl polymer and the N-terminus of FacVIIa.
  • the non-peptidyl is linked at one end to the immunoglobulin Fc region and at the other end to the N-terminus of the light chain of FacVIIa.
  • N-terminus used in the context of FacVIIa, is intended to encompass a region containing the N-terminus of FacVIIa.
  • the non-peptidyl polymer may be linked to the very N-terminal amino acid residue of FacVIIa or to an amino acid residue somewhat distant from the N-terminus so long as the FacVIIa complex retains the desired function.
  • FacVII is a single chain structure in which a light chain and a heavy chain are linked to each other before activation, only the N-terminus of the light chain is exposed outside. Converting FacVII to FacVIIa, cleavage between the arginine at position 152 and the isoleucine at position 153, exposes the active site of the heavy chain, with the isoleucine at position 153 accounting for the N-terminus of the heavy chain. Because the N-terminus of the heavy chain plays an important role in the activity of FacVIIa, the polymer must be linked to the N-terminus of the light chain, but not the heavy chain, so as to increase the titer.
  • PEG is linked to the N-terminus of an immunoglobulin Fc region and selectively coupled to the N-terminus of the light chain of FacVII to give a FacVII-PEG-immunoglobulin Fc complex. Afterwards, an additional activation process is carried out to complete the FacVIIa-PEG-immunoglobulin Fc complex.
  • the FacVIIa-PEG-immunoglobulin Fc complex prepared according to the present invention has a serum half-life of 60 hours, much longer than that of conventional therapeutic agents, and exhibits excellent blood coagulation effects in animal models, so that it can be prepared into long-acting FacVIIa formulations that retain excellent in vivo activity.
  • An immunoglobulin Fc region is a biodegradable polypeptide which can be metabolized in vivo, so that it can safely be used as a drug carrier.
  • an immunoglobulin Fc region is more advantageous in terms of production, purification and production yield because of its relatively smaller size compared to an entire immunoglobulin molecule.
  • amino acid sequence differs in one antibody to another, it can be expected that removal of highly heterogeneous Fab greatly increases homogeneity of substance and lower the likelihood of inducing blood antigenicity.
  • immunoglobulin Fc region refers to an immunoglobulin fragment that is devoid of the variable regions of light and heavy chains, the constant region 1 of the heavy chain (CH1) and the constant region 1 of the light chain (CL1), that is, a fragment comprised of the constant regions 2 and 3 of the heavy chain (CH2 and CH3).
  • the constant region of heavy chain may further comprise a hinge region.
  • the immunoglobulin Fc region of the present invention may be an extended Fc region which comprises a part of or the entirety of the constant region 1 of the heavy chain (CH1) and/or the constant region 1 of the light chain (CL1) in addition to the constant regions 2 and 3 of the heavy chain (CH2 and CH3) so long as it shows effects substantially identical or superior to those of the classical Fc region excluding only the variable regions of light and heavy chains of immunoglobulin. Further, it may be the region in which a considerably long part of amino acid sequence corresponding to CH2 and/or CH3 is deleted.
  • the immunoglobulin Fc region of the present invention may be composed of 1) CH1 domain, CH2 domain, CH3 domain and CH4 domain, 2) CH1 domain and CH2 domain, 3) CH1 domain and CH3 domain, 4) CH2 domain and CH3 domain, 5) a combination of one or more domains and an immunoglobulin hinge region (or a part of hinge region), or 6) a dimer of each constant domain of the heavy chain and the constant region of the light chain
  • the immunoglobulin Fc region of the present invention may include not only the wild-type Fc but its amino acid sequence mutant.
  • amino acid sequence mutant refers to an amino acid sequence that is different from the wild-type as a result of deletion, insertion, conserved or non-conserved substitution of one or more amino acid residues, or a combination thereof. For instance, amino acid residues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331in IgG Fc, known to be important for linkage, may be used as the sites suitable for modification.
  • Various derivatives such as those prepared by removing the sites of disulfide bonds, removing several N-terminal amino acids from native Fc, or adding methionine to the N-terminus of native Fc, may be available.
  • complement fixation sites e.g., C1q fixation sites, or ADCC sites may be eliminated to remove the effector function from the native Fc region.
  • the techniques of preparing amino acid sequence mutants of the immunoglobulin Fc region are disclosed in International Patent Publication Nos. WO 97/34631 and WO 96/32478 and so forth.
  • Amino acid substitutions in a protein or peptide molecule that do not alter the activity of the molecule are well known in the art (H.Neurath, R.L.Hill, The Proteins, Academic Press, New York,197 9). The most common substitutions occur between amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.
  • amino acids may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation.
  • Fc derivatives exhibit the same biological activity as that of the wild-type, but have improved structural stability when subjected to heat and pH.
  • Fc regions may be obtained as native Fc regions from humans or animals such as cow, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., or as recombinant or derived Fc regions from transformed animal cells or microorganisms.
  • Native Fc regions may be obtained by protease digestion of the gamut of immunoglobulins isolated from human or animal samples. Immunoglobulins are cleaved into Fab and Fc by papain and into pF'c and F(ab')2 by pepsin, followed by size-exclusion chromatography to separate Fc or pF'c therefrom.
  • a recombinant human Fc region obtained from a microorganism is preferred.
  • the immunoglobulin Fc region useful in the present invention may be glycosylated to the same extent as or to a higher and lesser extent than the native form or may be deglycosylated. Increased or decreased glycosylation or deglycosylation of the immunoglobulin region may be achieved by typical methods, for example, by using a chemical method, an enzymatic method or a genetic engineering method using a microorganism.
  • the complement (C1q) binding of an immunoglobulin Fc region becomes significantly decreased and it has reduced or no antibody-dependent cytotoxicity or complement-dependent cytotoxicity, so that it does not induce unnecessary immune responses in vivo.
  • deglycosylated or aglycosylated immunoglobulin Fc regions are more consistent with the purpose of being as drug carriers.
  • deglycosylation is intended to mean the enzymatic removal of sugars from an Fc region.
  • amino acid sequence when used in conjunction with an Fc region, means an Fc region free of sugars, expressed from prokaryotes, preferably from E. coli.
  • the immunoglobulin Fc region may originate from humans or animals such as cows, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., and preferably is of human origin.
  • the immunoglobulin Fc region may be derived from IgG, IgA, IgD, IgE, IgM, or combinations or hybrids thereof.
  • the Fc region is derived from IgG or IgM, which are the most abundant ones in human blood, and most preferably from IgG, which is known to improve the serum half-life of ligand-binding proteins.
  • a dimer or multimer may be formed from two or more fragments selected from the group consisting of IgG1 Fc, IgG2 Fc, IgG3 Fc and IgG4 Fc fragments.
  • hybrid means that sequences encoding two or more immunoglobulin Fc fragments of different origin are present in a single-chain immunoglobulin Fc fragment.
  • domain hybrids may be composed of one to four domains selected from the group consisting of CH1, CH2, CH3 and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc, and may include the hinge region.
  • IgG is divided into the IgG1, IgG2, IgG3 and IgG4 subclasses, and the present invention may include combinations or hybrids thereof. Preferred are the IgG2 and IgG4 subclasses, and most preferred is the Fc region of IgG4 rarely having effector functions such as CDC (Complement Dependent Cytotoxicity).
  • the immunoglobulin Fc region most suitable as the drug carrier of the present invention is a human IgG4-derived aglycosylated Fc region.
  • the human-derived Fc region is more preferable than a non-human derived Fc region, which may act as an antigen in the human body and cause undesirable immune responses such as the production of a new antibody against the antigen.
  • non-peptidyl polymer refers to a biocompatible polymer comprised of at least two repeating units which are held together by any covalent bond other than a peptide bond.
  • the non-peptidyl polymer may have two or three terminal functional groups.
  • a useful non-peptidyl polymer may be selected from polyethylene glycol, poly propylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers such as PLA (poly (lactic acid) and PLGA (poly (lactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, and a combination thereof.
  • PLA poly (poly (lactic acid) and PLGA (poly (lactic-glycolic acid)
  • lipid polymers chitins, hyaluronic acid, and a combination thereof.
  • the most preferred is polyethylene glycol.
  • Their derivatives are well known in the art and derivatives which can be readily prepared using a method known in the art are also within the scope of the present invention.
  • any non-peptidyl polymer may be used in the present invention, without limitation.
  • the non-peptidyl polymer ranges in molecular weight from 1 to 100 kDa and preferably from 1 to 20 kDa.
  • the non-peptidyl polymer which is linked to the immunoglobulin Fc region may be not only an individual polymer but a combination of different polymers.
  • the non-peptidyl polymer useful in the present invention has functional groups which are coupled to an immunoglobulin Fc region and a protein drug.
  • the non-peptidyl polymer discussed above has two or three termini.
  • the functional group is preferably selected from the group consisting of aldehyde, propion aldehyde, butyl aldehyde, maleimide, and succinimide derivative.
  • succinimide its derivatives including, succinimidyl propionate, hydroxy succinimidyl, succinimidyl carboxymethyl or succinimidyl carbonate may also be used.
  • the non-peptidyl polymer has aldehyde functional groups at its both ends, it can be effectively linked at both ends to a physiologically active polypeptide and an immunoglobulin, respectively, with minimal non-specific reactions therebetween.
  • the final products produced by reductive alkylation via an aldehyde bond are much more stable than those linked via an amide bond.
  • An aldehyde functional group specifically reacts with an amino terminus at low pH, and can form a covalent bond with a lysine residue at high pH, e.g., a pH of 9.0.
  • the two or three terminal functional groups of the non-peptidyl polymer may be the same or different.
  • the non-peptidyl polymer may have a maleimide group at one end and an aldehyde group, a propionaldehyde group, or a butyl aldehyde group at the other or another end.
  • the hydroxy group may be activated into the above-mentioned functional groups before being used in the present invention.
  • commercially available poly(ethylene glycol) with modified functional groups may be used to prepare the protein complex of the present invention.
  • the present invention provides a pharmaceutical composition for blood coagulation, comprising the FacVIIa complex.
  • the present invention provides a pharmaceutical composition for the treatment of blood coagulation-related diseases including hemophilia, bleeding, acute intracerebral hemorrhage, wounds and FacVII deficiency.
  • blood coagulation-related diseases including hemophilia, bleeding, acute intracerebral hemorrhage, wounds and FacVII deficiency.
  • administration means the introduction of a predetermined amount of a substance into a patient by a certain suitable method. So long as it is able to induce the complex to reach a target tissue, any route of administration may be used.
  • modes of administration are contemplated, including intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, topically, intranasally, intrapulmonarily and intrarectally, but the present invention is not limited to these modes of administration.
  • active ingredients of the composition for oral administration should be coated or formulated for protection against degradation in the stomach.
  • the composition of the present invention may be administered in an injectable form.
  • the pharmaceutical composition of the present invention may be administered using a certain apparatus capable of transporting the active ingredients into a target cell.
  • the pharmaceutical composition comprising the complex according to the present invention may comprise a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may include binders, lubricants, disintegrators, excipients, solubilizers, dispersing agents, stabilizers, suspending agents, coloring agents and odoriferous substances.
  • the pharmaceutically acceptable carrier may include buffering agents, preserving agents, analgesics, solubilizers, isotonic agents and stabilizers.
  • the pharmaceutically acceptable carrier may include bases, excipients, lubricants and preserving agents.
  • the pharmaceutical composition of the present invention may be formulated into a variety of dosage forms in combination with the aforementioned pharmaceutically acceptable carriers.
  • the pharmaceutical composition may be formulated into tablets, troches, capsules, elixirs, suspensions, syrups or wafers.
  • the pharmaceutical composition may be formulated into a unit dosage form, such as an ampule in single-dose dosage form or a multidose container.
  • the pharmaceutical composition may also be formulated into solutions, suspensions, tablets, pills, capsules and long-acting preparations.
  • Examples of carriers, excipients and diluents suitable for the pharmaceutical formulations include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils.
  • the pharmaceutical formulations may further include fillers, anti-coagulating agents, lubricants, humectants, odoriferous substances, and antiseptics.
  • a dosage of the pharmaceutical composition of the present invention may be determined by the type of the drug which is the active component as well as by several related factors including the type of disease to be treated, administration route, the patient's age, gender, weight and severity of the illness. Since the pharmaceutical composition of the present invention has a very long duration of action in vivo and a high titer, it has the advantage of greatly reducing the frequency of administration of the pharmaceutical drugs.
  • the present invention provides a method for treating a blood coagulation-related disease, which comprises administering the FacVIIa complex or the pharmaceutical composition of this invention to a subject in needs thereof.
  • the disease is one caused by the insufficient coagulation of blood, and may include, but is not limited to, hemophilia, bleeding, acute intracerebral hemorrhage, wounds, and FacVII deficiency.
  • the subject may be mammals including, but not limited to, humans, mice, pigs, cows, dogs, sheep, etc. with a preference for humans.
  • the FacVIIa complex, the composition and the administration are as described above.
  • the present invention provides a method for preparing a FacVIIa complex, comprising:
  • FacVII covalently linking FacVII to another end of the non-peptidyl polymer of the isolated conjugate to afford a FacVII complex in which the non-peptidyl polymer is linked at one end to the immunoglobulin Fc region and at another end to FacVII;
  • step (3) (4) activating the FacVII complex of step (3) into a FacVIIa complex in which FacVIIa is linked to the immunoglobulin Fc region via the non-peptidyl polymer.
  • the present invention provides a method for preparing a FacVIIa complex, comprising:
  • FacVII covalently linking FacVII to another end of the non-peptidyl polymer of the conjugate to form a FacVII complex in which the non-peptidyl polymer is linked at one end to the immunoglobulin Fc region and at another end to FacVII;
  • step (3) (4) activating the FacVII complex of step (3) into a FacVIIa complex in which FacVIIa is linked to the immunoglobulin Fc region via the non-peptidyl polymer.
  • the present invention provides a method for preparing a FacVIIa complex, comprising:
  • step (3) (4) activating the FacVII complex of step (3) into a FacVIIa complex in which FacVIIa is linked to the immunoglobulin Fc region via the non-peptidyl polymer.
  • the FacVII complex is attached to an anion exchange column and activated into the FacVIIa complex by on-column activation (autoactivation).
  • the FacVII is linked preferably at its N-terminus to the non-peptidyl polymer.
  • the N-terminus mentioned above is from the light chain of FacVII.
  • FacVII is native FacVII, or a FacVII agonist, precursor, derivative, fragment or variant. Most preferred is the native FacVII.
  • the FacVIIa is native FacVIIa, or a FacVIIa agonist, precursor, derivative, fragment or variant. Most preferred is native FacVIIa.
  • PLA poly (poly (lactic acid)
  • PLGA poly (lactic-glycolic acid)
  • lipid polymers chitins, and hyaluronic acid.
  • the most preferred is polyethylene glycol.
  • the non-peptidyl polymer has an aldehyde derivative as a terminal group and more preferably has aldehyde functional groups at three termini.
  • An immunoglobulin Fc was pegylated at the N terminus with 5K PropionALD(3) PEG (PEG with three terminal propionaldehyde groups, NOF, Japan).
  • 6 mg/mL immunoglobulin Fc was reacted with PEG at 4°C for 4.5 hrs with the molar ratio of immunoglobulin Fc to PEG set at 1:2.
  • the reaction was performed in 100 mM potassium phosphate buffer at pH 6.0 in the presence of 20mM SCB (NaCNBH 3 ) as a reducing agent.
  • the reaction mixture was loaded onto SOURCE Q (LRC25 85ml, Pall Corporation) to purify the mono-pegylated immunoglobulin Fc.
  • FVII was coupled with the immunoglobulin Fc-5K PEG at a molar ratio of 1 : 10 (FVII : immunoglobulin Fc-5K PEG) at 4°C for 18 hrs, with the total protein concentration set to 20 mg/mL.
  • the coupling reaction was performed in 100mM potassium phosphate at pH 6.0 in the presence of 20 mM SCB as a reducing agent.
  • the coupling reaction mixture was purified by passing it through two columns.
  • SOURCE Q LRC25 85ml, Pall Corporation
  • the immunoglobulin Fc-3 arm PEG-FVII was reloaded onto SOURCE Q, followed by pouring a mobile phase containing 1.75 mM calcium ion on the column for 6 hours. Elution was carried out with 35 mM calcium ions to afford immunoglobulin Fc-3 arm PEG-FVIIa.
  • FVII FVII
  • 20K mPEG butylaldehyde Naktar, USA
  • 5 mg/mL FVII was reacted with PEG at 4°C for 10 hrs with the molar ratio of FVII to 20K PEG set at 1:5.
  • the reaction was performed in 100 mM sodium acetate buffer at pH 5.0 in the presence of 20mM SCB (NaCNBH 3 ) as a reducing agent.
  • the mono-pegylated FVII was purified through RESOURCE Q (1ml, prepacked, GE Healthcare).
  • FVII was pegylated at a lysine residue with 20k mPEG SPA (Nektar, USA).
  • 20k mPEG SPA Naktar, USA
  • 3 mg/mL FVII was reacted with 20k PEG at room temperature for 3 hrs with the molar ratio of FVII to 20k PEG set at 1:5.
  • the reaction was performed in 100 mM sodium phosphate buffer at pH 8.0.
  • the mono-pegylated FVII was purified through RESOURCE Q (1 ml, prepacked, GE Healthcare). Given a salt gradient of 1 M NaCl in 20mM Tris (pH 7.5), the column eluted multi-pegylated FVII, mono-pegylated FVII and FVII in that order.
  • FVIIa and immunoglobulin Fc-PEG-FVIIa were each intravenously injected at a dose of 100 ⁇ g/kg into normal SD rats, followed by ELISA analysis to obtain serum levels.
  • a blood sample was collected at 0.25, 0.5, 1, 2, 5, 10, 24, and 48hrs for the FVIIa-administered rats, and at 0.25, 0.5, 1, 2, 5, 10, 24, 48, 72, 96 and 120 hrs for the immunoglobulin Fc-PEG-FVIIa-administered rats.
  • the blood samples were collected in tubes with sodium citrate to prevent coagulation, and centrifuged for 5 min using an Eppendorf high-speed micro centrifugator to separate serum. Serum protein levels were measured by ELISA (IMUBIND, Factor VIIa ELISA Kit, American diagnostic inc.) using antibodies specific to FVIIa.
  • Tmax accounts for the time taken to reach the maximal serum concentration of a drug
  • T1/2 for the serum half-life of a drug
  • MRT mean residence time
  • immunoglobulin Fc-PEG-FVIIa was observed to have a great serum half-life of as long as about 60 hrs.
  • Activity assay was performed according to the instructions described in "2.7.10. ASSAY OF HUMAN COAGULATION FACTOR VII" of the European Pharmacopoeia.
  • FX was activated into FXa by treatment with dilutions of Novoseven, FVIIa and immunoglobulin Fc-PEG-FVIIa at various concentrations and S-2765 used as a substrate was hydrolyzed into a peptide and pNA, a chromophoric group, by the FXa.
  • the yellow color of the hydrolyzed pNA was used to measure absorbance at 405 nm on an ELIAS reader.
  • a dose responsive curve and EC50 values were determined using the measured absorbance and the treated concentrations of the drug.
  • immunoglobulin Fc-PEG-FacVIIa was observed to have an EC50 of 50.72 ng/mL, which is 27-fold higher than that of Novoseven [FIG. 2].
  • FVIIa and immunoglobulin Fc-PEG-FVIIa were assayed for in vivo FacVIIa activity depending on the administration of test drugs in SD rats pre-treated with warfarin.
  • Novoseven was used which is a recombinant form of FVIIa commercially available from Novo Nordisk, which is applied to the treatment for bleeding hemophilias and the hemostasis of patients under a surgical operation.
  • Warfarin which acts to inhibit the gamma-carboxylation of vitamin K-dependent coagulation factors such as Factor II, IX, X and VII, was administered to SD rats 24 hrs ahead, after which Novoseven, FVIIa, and immunoglobulin Fc-PEG-FVIIa were individually intravenously injected at dosages of 250 ⁇ g to the SD rats.
  • One mL of blood was sampled from the jugular vein at 0.4, 4, 24, and 48 hrs after the intravenous injection, using tubes containing sodium citrate.
  • FVII activity (%) from the isolated serum was measured using ACL9000 (Werfen group).
  • the FacVIIa complex of the present invention guarantees the in vivo activity of FacVIIa and significantly enhances the serum half live of FacVIIa, so that it is useful for developing long-acting FacVIIa formulations which can be in compliance with the role behaviors of patients whose blood do not coagulate.

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Abstract

L'invention concerne un complexe contenant un facteur de coagulation sanguine, dans lequel FacVIIa, un polymère non peptidique et une région Fc d'immunoglobuline sont liés par des liaisons covalentes, et son utilisation. Le complexe de FacVIIa garantit l'activité in vivo de FacVIIa et augmente significativement la demi-vie sérique de FacVIIa, de sorte qu'il est utile pour développer des préparations contenant FacVIIa à longue durée d'action, qui sont mieux acceptées par les patients dont le sang ne coagule pas.
PCT/KR2011/004796 2010-06-30 2011-06-30 Formation d'un complexe avec le facteur viia au moyen d'un fragment d'immunoglobuline WO2012002745A2 (fr)

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JP2013518254A JP2013533875A (ja) 2010-06-30 2011-06-30 免疫グロブリン断片を用いた第７因子（ＦａｃｔｏｒＶＩＩａ）薬物結合体
CN2011800325807A CN103025358A (zh) 2010-06-30 2011-06-30 使用免疫球蛋白片段的因子viia复合物
US13/807,572 US20130095090A1 (en) 2010-06-30 2011-06-30 Factor viia complex using an immunoglobulin fragment
EP11801151.9A EP2588142A2 (fr) 2010-06-30 2011-06-30 Formation d'un complexe avec le facteur viia au moyen d'un fragment d'immunoglobuline

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Cited By (2)

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WO2015154139A1 (fr) * 2014-04-11 2015-10-15 Csl Limited Protéine de facteur fviia à demi-vie prolongée pour la prévention et le traitement d'hémorragie, et leurs régimes posologiques
US11626188B2 (en) 2016-01-21 2023-04-11 Protein Dynamic Solutions, Inc. Method and system for spectral data analysis

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ES2372495T3 (es) * 2003-11-13 2012-01-20 Hanmi Holdings Co., Ltd Método para la producción en masa de la región constante de inmunoglobulina.
CN103397009B (zh) * 2013-08-16 2015-06-03 安源生物科技(上海)有限公司 改良型人凝血因子FVII-Fc融合蛋白及其制备方法与用途
KR101892687B1 (ko) 2017-05-18 2018-08-28 세종대학교산학협력단 하이드라진 연료전지용 전극과 이를 포함하는 막 전극 접합체 및 연료전지
CN111849945A (zh) * 2019-04-25 2020-10-30 正大天晴药业集团股份有限公司 人凝血因子VIIa的纯化方法

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US20090285780A1 (en) * 2006-05-24 2009-11-19 Chyi Lee Peg linker compounds and biologically active conjugates thereof
US20090305967A1 (en) * 2005-08-19 2009-12-10 Novo Nordisk A/S Glycopegylated factor vii and factor viia

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ES2574581T3 (es) * 2003-08-14 2016-06-20 Novo Nordisk Health Care Ag Composición farmacéutica líquida acuosa de polipéptidos de tipo Factor VII
JP2007509843A (ja) * 2003-10-07 2007-04-19 ノボ ノルディスク ヘルス ケア アクチェンゲゼルシャフト 第ＶＩＩ／ＶＩＩａ因子活性を有するハイブリッド分子
JP5570809B2 (ja) * 2006-09-01 2014-08-13 ノボ ノルディスク ヘルス ケア アーゲー 修飾タンパク質
JP5563572B2 (ja) * 2008-07-23 2014-07-30 ハンミ サイエンス カンパニー リミテッド 三末端官能基を有する非ペプチド性重合体を用いた生理活性ポリペプチド薬物結合体

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US20060269553A1 (en) * 2003-11-13 2006-11-30 Hanmi Pharm. Ind. Co., Ltd. Protein complex using an immunoglobulin fragment and method for the preparation thereof
US20090305967A1 (en) * 2005-08-19 2009-12-10 Novo Nordisk A/S Glycopegylated factor vii and factor viia
US20090285780A1 (en) * 2006-05-24 2009-11-19 Chyi Lee Peg linker compounds and biologically active conjugates thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015154139A1 (fr) * 2014-04-11 2015-10-15 Csl Limited Protéine de facteur fviia à demi-vie prolongée pour la prévention et le traitement d'hémorragie, et leurs régimes posologiques
JP2017513831A (ja) * 2014-04-11 2017-06-01 シーエスエル、リミテッド 出血の予防および処置のための半減期延長型（ｈａｌｆ−ｌｉｆｅ ｅｘｔｅｎｄｅｄ）第ｆｖｉｉａ因子タンパク質、ならびに、その投与レジメン
US11626188B2 (en) 2016-01-21 2023-04-11 Protein Dynamic Solutions, Inc. Method and system for spectral data analysis

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